Ryanodine Receptor Open Probability Research Articles

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: I. Transverse-axial tubule variation.

Intracellular calcium (Ca2+ ) cycling is tightly regulated in the healthy heart ensuring effective contraction. This is achieved by transverse (t)-tubule membrane invaginations that facilitate close coupling of key Ca2+ -handling proteins such as the L-type Ca2+ channel and Na+ -Ca2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca2+ store. Although less abundant and regular than in the ventricle, t-tubules also exist in atrial myocytes as a network of transverse invaginations with axial extensions known as the transverse-axial tubule system (TATS). In heart failure and atrial fibrillation, there is TATS remodelling that is associated with aberrant Ca2+ -handling and Ca2+ -induced arrhythmic activity; however, the mechanism underlying this is not fully understood. To address this, we developed a novel 3D human atrial myocyte model that couples electrophysiology and Ca2+ -handling with variable TATS organization and density. We extensively parameterized and validated our model against experimental data to build a robust tool examining TATS regulation of subcellular Ca2+ release. We found that varying TATS density and thus the localization of key Ca2+ -handling proteins has profound effects on Ca2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca2+ concentration through decreased Ca2+ extrusion. This elevated Ca2+ increases RyR open probability causing spontaneous Ca2+ releases and the promotion of arrhythmogenic waves (especially in the cell interior) leading to voltage instabilities through delayed afterdepolarizations. In summary, the present study demonstrates a mechanistic link between TATS remodelling and Ca2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca2+ and voltage instabilities through reduced Na+ -Ca2+ exchanger-mediated Ca2+ removal, cleft Ca2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca2+ releases are larger and more frequent in the cell interior than at the periphery. Our work provides mechanistic insight into how atrial TATS remodelling can lead to Ca2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease.

Cardiac Alternans Occurs through the Synergy of Voltage- and Calcium-Dependent Mechanisms.

Cardiac alternans is characterized by alternating weak and strong beats of the heart. This signaling at the cellular level may appear as alternating long and short action potentials (APs) that occur in synchrony with alternating large and small calcium transients, respectively. Previous studies have suggested that alternans manifests itself through either a voltage dependent mechanism based upon action potential restitution or as a calcium dependent mechanism based on refractoriness of calcium release. We use a novel model of cardiac excitation-contraction (EC) coupling in the rat ventricular myocyte that includes 20,000 calcium release units (CRU) each with 49 ryanodine receptors (RyR2s) and 7 L-type calcium channels that are all stochastically gated. The model suggests that at the cellular level in the case of alternans produced by rapid pacing, the mechanism requires a synergy of voltage- and calcium-dependent mechanisms. The rapid pacing reduces AP duration and magnitude reducing the number of L-type calcium channels activating individual CRUs during each AP and thus increases the population of CRUs that can be recruited stochastically. Elevated myoplasmic and sarcoplasmic reticulum (SR) calcium, [Ca2+]myo and [Ca2+]SR respectively, increases ryanodine receptor open probability (Po) according to our model used in this simulation and this increased the probability of activating additional CRUs. A CRU that opens in one beat is less likely to open the subsequent beat due to refractoriness caused by incomplete refilling of the junctional sarcoplasmic reticulum (jSR). Furthermore, the model includes estimates of changes in Na+ fluxes and [Na+]i and thus provides insight into how changes in electrical activity, [Na+]i and sodium-calcium exchanger activity can modulate alternans. The model thus tracks critical elements that can account for rate-dependent changes in [Na+]i and [Ca2+]myo and how they contribute to the generation of Ca2+ signaling alternans in the heart.

X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin.

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.

Early Afterdepolarizations and Alternans are the Underlying Mechanism to Cause Arrhythmogenic Disorder in the Mutant Calsequestrin 2 (CASQ2)

Calsequestrin 2 (CASQ2) is high capacity and low-affinity Ca2+-binding protein expressed in the sarcoplasmic reticulum (SR) of cardiac myocyte. Mutation that occurs in CASQ2 expressing gene have been linked to arrhythmia and possibly sudden cardiac death (SCD) with acute emotional stress or exercise. This inherited genetic disorder is known as catecholamine polymorphic ventricular tachycardia (CPVT). Understanding the subcellular mechanisms of CPVT is experimentally challenging because the arrhythmias are rare and the development of SCD are even rarer. The underlying causes of CPVT have been described as delayed afterdepolarizations (DADs), early afterdepolarizations (EADs) or Ca2+ signaling alternans. To gain insight into the nature of this rare disease, we have developed a local control stochastic model of the cardiac myocyte to investigate how the mutant CASQ2s may be responsible for the development of an arrhythmogenic episode under the condition of beta-adrenergic stimulation or in the pauses afterward. We choose a long action potential model to contrast with our previous studies with the short action potential rat model. Experimental observation indicate that the mutation is accompanied by increased ryanodine receptor open probability and increased junctional sarcoplasmic reticulum volume. The model simulates EADs at low pacing frequency. At high pacing frequency, alternans occurs not due to spark restitution as seen in the rat model, but by the accumulated inactivation of the sodium channel. These alternans do not require changes in jSR volume which were important in the spark restitution-based mechanism. The model offers novel insights into how mechanisms can vary between a long- and short- action potential models of arrhythmia.

Control of Neuronal Ryanodine Receptor-Mediated Calcium Signaling by Calsenilin.

Calsenilin is a calcium ion (Ca2+)-binding protein involved in regulating the intracellular concentration of Ca2+, a second messenger that controls multiple cellular signaling pathways. The ryanodine receptor (RyR) amplifies Ca2+ signals entering the cytoplasm by releasing Ca2+ from endoplasmic reticulum (ER) stores, a process termed calcium-induced calcium release (CICR). Here, we describe a novel mechanism, in which calsenilin controls the activity of neuronal RyRs. We show calsenilin co-localized with RyR2 and 3 in the ER of mouse hippocampal and cortical neurons using immunocytochemistry. The underlying protein-protein interaction between calsenilin and the RyR was determined in mouse central nervous system (CNS) neurons using immunoprecipitation studies. The functional relevance of this interaction was assayed with single-channel electrophysiology. At low physiological Ca2+ concentrations, calsenilin binding to the cytoplasmic face of neuronal RyRs decreased the RyR's open probability, while calsenilin increased the open probability at high physiological Ca2+ concentrations. This novel molecular mechanism was studied further at the cellular level, where faster release kinetics of caffeine-induced Ca2+ release were measured in SH-SY5Y neuroblastoma cells overexpressing calsenilin. The interaction between calsenilin and neuronal RyRs reveals a new regulatory mechanism and possibly a novel pharmacological target for the control of Ca2+ release from intracellular stores.

Arrhythmogenic Delayed Afterdepolarizations Are Promoted by Severe Hypothermia But Not Therapeutic Hypothermia.

Severe hypothermia (SH) is known to be arrhythmogenic, but the effect of therapeutic hypothermia (TH) on arrhythmias is unclear. It is hypothesized that susceptibility to Ca-mediated arrhythmia triggers would be increased only by SH.Methods and Results:Spontaneous Ca release (SCR) and resultant delayed afterdepolarizations (DADs) were evaluated by optical mapping in canine wedge preparations during normothermia (N, 36℃), TH (32℃) or SH (28℃; n=8 each). The slope (amplitude/rise time) of multicellular SCR (mSCR) events, a determinant of triggered activity, was suppressed in TH (24.4±3.4%/s vs. N: 41.5±6.0%/s), but significantly higher in SH (96.3±8.1%/s) producing higher amplitude DADs in SH (35.7±1.6%) and smaller in TH (5.3±1.0% vs. N: 10.0±1.1%, all P<0.05). Triggered activity was only observed in SH. In isolated myocytes, sarcoplasmic reticulum (SR) Ca release kinetics slowed in a temperature-dependent manner, prolonging Ca transient rise time [33±3 (N) vs. 50±6 (TH) vs. 88±12 ms (SH), P<0.05], which can explain the decreased mSCR slope and DAD amplitude in TH. Although the SR Ca content was similar in TH and SH, Ca spark frequency was markedly increased only in SH, suggesting that increased ryanodine receptor open probability could explain the increased triggered activity during SH. Temperature dependence of Ca release can explain susceptibility to Ca-mediated arrhythmia triggers in SH. This may therefore explain the increased risk of lethal arrhythmia in SH, but not during TH.

Long-Lasting Sparks: Multi-Metastability and Release Competition in the Calcium Release Unit Network.

Calcium (Ca) sparks are elementary events of biological Ca signaling. A normal Ca spark has a brief duration in the range of 10 to 100 ms, but long-lasting sparks with durations of several hundred milliseconds to seconds are also widely observed. Experiments have shown that the transition from normal to long-lasting sparks can occur when ryanodine receptor (RyR) open probability is either increased or decreased. Here, we demonstrate theoretically and computationally that long-lasting sparks emerge as a collective dynamical behavior of the network of diffusively coupled Ca release units (CRUs). We show that normal sparks occur when the CRU network is monostable and excitable, while long-lasting sparks occur when the network dynamics possesses multiple metastable attractors, each attractor corresponding to a different spatial firing pattern of sparks. We further highlight the mechanisms and conditions that produce long-lasting sparks, demonstrating the existence of an optimal range of RyR open probability favoring long-lasting sparks. We find that when CRU firings are sparse and sarcoplasmic reticulum (SR) Ca load is high, increasing RyR open probability promotes long-lasting sparks by potentiating Ca-induced Ca release (CICR). In contrast, when CICR is already strong enough to produce frequent firings, decreasing RyR open probability counter-intuitively promotes long-lasting sparks by decreasing spark frequency. The decrease in spark frequency promotes intra-SR Ca diffusion from neighboring non-firing CRUs to the firing CRUs, which helps to maintain the local SR Ca concentration of the firing CRUs above a critical level to sustain firing. In this setting, decreasing RyR open probability further suppresses long-lasting sparks by weakening CICR. Since a long-lasting spark terminates via the Kramers’ escape process over a potential barrier, its duration exhibits an exponential distribution determined by the barrier height and noise strength, which is modulated differently by different ways of altering the Ca release flux strength.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

Cardiac Alternans Occurs through the Synergy of Voltage- and Calcium-Dependent Mechanisms

Cardiac alternans is characterized by alternating weak and strong beats of the heart. This signaling at the cellular level may appear as alternating long and short action potentials (APs) that occur in synchrony with alternating large and small calcium transients, respectively. Previous studies have suggested that alternans manifests itself through either a voltage dependent mechanism based upon action potential restitution or as a calcium dependent mechanism based on refractoriness of calcium release. We use a novel model of cardiac excitation-contraction (EC) coupling in the rat ventricular myocyte that includes 20,000 calcium release units (CRU) each with 49 ryanodine receptors and 7 L-type calcium channels that are all stochastically-gated. The model suggests that at the cellular level in the case of alternans produced by rapid pacing, the mechanism requires a synergy of voltage- and calcium-dependent mechanisms. The rapid pacing reduces AP duration and magnitude reducing the number of L-type calcium channels activating individual CRUs during each AP and thus increases the population of CRUs that can be recruited stochastically. Elevated myoplasmic and sarcoplasmic reticulum (SR) calcium, [Ca2+]i and [Ca2+]SR respectively, increases ryanodine receptor open probability (Po) according to our model used in this simulation and this increased the probability of activating additional CRUs. A CRU that opens in one beat is less likely to open the subsequent beat due to refractoriness caused by incomplete refilling of the junctional sarcoplasmic reticulum (jSR). Furthermore, the model includes estimates of changes in Na+ fluxes and [Na+]i and thus provides insight into how changes in electrical activity, [Na+]i and sodium-calcium exchanger activity can modulate alternans. The model thus tracks critical elements that can account for rate-dependent changes in [Na+]i and [Ca2+]i and how they contribute to the generation of Ca2+ signaling alternans in heart.

Calcium in the heart: from physiology to disease

Contraction of the heart results from an increase of cytoplasmic Ca(2+) concentration ([Ca(2+)]i), the so-called systolic Ca(2+) transient. Most of this results from the release of Ca(2+) from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). In turn, the amplitude of this Ca(2+) transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca(2+) release. The amplitude of the Ca(2+) transient is a steep function of SR Ca, resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism, in which an increase of SR Ca content increases the size of the Ca(2+) transient, resulting in a decrease of Ca(2+) influx on the L-type Ca(2+) current and an increase of efflux through Na(+)-Ca(2+) exchange. Changing the activity of any of the Ca(2+)-cycling proteins will change the steady-state SR Ca content. This feedback mechanism has many consequences, including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca(2+) transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca(2+) waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia, which is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Calcium waves occur only when the SR Ca content exceeds a threshold level. Our data show that the threshold is reduced by the RyR mutation and that the adrenergic stimulation increases SR Ca content.

Simultaneous Measurement of Cytoplasmic and SR Calcium during Modulation of Ryanodine Receptor Open Probability in Dog Ventricular Myocytes

We have previously found that, in rat ventricular myocytes, increasing ryanodine receptor open probability using low concentrations of caffeine had no maintained effect on the amplitude of systolic Ca. On application of caffeine, following an initial increase, systolic Ca returned to control levels in around 20 s and it was argued that this was due to a concurrent decrease in SR Ca. In the present study, we sought to obtain direct evidence for the involvement of a decrease of SR Ca in this transient response.SR Ca was measured directly with Mag-Fura-2. Application of 0.5 mM caffeine initially caused a 197 % increase in the amplitude of systolic Ca. This was associated with a 874 % increase in the amplitude of SR Ca loss and a 328 and 178 % increase in the rate of systolic cytoplasmic Ca removal and SR Ca replenishment respectively. In sustained caffeine exposure, all these parameters returned to levels comparable to control, typically within 1 - 2 beats. During caffeine exposure, SR Ca content rapidly decreased within 1 - 2 beats to a new steady state level. All measured parameters recovered to control levels on removal of caffeine.These data show, on RyR potentiation, following the initial increase, the secondary decrease of systolic Ca is due to a decrease in SR Ca.

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Is ryanodine receptor a calcium or magnesium channel? Roles of K+ and Mg2+ during Ca2+ release

The ryanodine receptor (RyR) is a poorly selective channel that mediates Ca2+ release from intracellular Ca2+ stores. How RyR's selectivity between the physiological cations K+, Mg2+, and Ca2+ affects single-channel Ca2+ current amplitude is examined using a recent model of RyR permeation. It is found that K+ provides the vast majority of the countercurrent (through RyR itself) that is needed to prevent the sarcoplasmic reticulum (SR) membrane potential from changing and stopping Ca2+ release. Moreover, intra-pore competition between Ca2+ and Mg2+ defines single RyR Ca2+ current amplitude. Since both [Mg2+] and [Ca2+]SR can change during pathophysiological conditions, the RyR unitary Ca2+ current amplitude during Ca2+ release may change significantly due to this Ca2+/Mg2+ competition. Compared to the classic action of Mg2+ on RyR open probability, these Ca2+ current amplitude changes have as large or larger effects on overall RyR Ca2+ mobilization. A new aspect of RyR divalent versus monovalent selectivity is also identified where this kind of selectivity decreases as divalent concentration increases.

Construction of Calcium Release Sites in Cardiac Myocytes

Local character of calcium release in cardiac myocytes, as defined by confocal recordings of calcium sparks, implies independent activation of individual calcium release sites based on ryanodine receptor (RyR) channel recruitment. We constructed virtual calcium release sites (vCRSs) composed of a variable number of RyR channels distributed in clusters in accordance with the experimentally observed cluster size distribution. The vCRSs consisted either of a single virtual calcium release unit (vCRU), in which all clusters shared a common dyadic space, or of multiple virtual calcium release units (CRUs) containing one cluster each and having separate dyadic spaces. We explored the stochastic behavior of vCRSs to understand the activation and recruitment of RyRs during calcium sparks. RyRs were represented by the published allosteric gating model that included regulation by cytosolic Ca2+ and Mg2+. The interaction of Mg2+ with the RyR Ca2+-binding sites and the refractory period of vCRSs were optimized to accord with the experimentally observed calcium dependence of calcium spark frequency. The Mg2+-binding parameters of RyRs that provided the best description of spark frequency depended on the number of RyRs assembled in the vCRSs. Adequate inhibitory effect of Mg2+ on the calcium dependence of RyR open probability was achieved if the vCRSs contained at least three clusters. For the distribution of the number of open RyRs in evoked calcium sparks to correspond to the experimentally observed distribution of spark calcium release fluxes, at least three clusters had to share a common virtual CRU, in which ∼3 RyRs open to form an average spark. These results reconcile the small cluster size and stochastic placement of RyRs in the release sites with the estimates of the amount of RyR protein, volume density of calcium release sites, and the size of calcium release sites in rat cardiac myocytes.

Spontaneous calcium oscillations during diastole in the whole heart: the influence of ryanodine reception function and gap junction coupling

Triggered arrhythmias due to spontaneous cytoplasmic calcium oscillations occur in a variety of disease conditions; however, their cellular mechanisms in tissue are not clear. We hypothesize that spontaneous calcium oscillations in the whole heart are due to calcium release from the sarcoplasmic reticulum and are facilitated by calcium diffusion through gap junctions. Optical mapping of cytoplasmic calcium from Langendorff perfused guinea pig hearts (n = 10) was performed using oxygenated Tyrode's solution (in mM): 140 NaCl, 0.7 MgCl, 4.5 KCl, 5.5 dextrose, 5 HEPES, and 5.5 CaCl₂ (pH 7.45, 34°C). Rapid pacing was used to induce diastolic calcium oscillations. In all preparations, pacing-induced multicellular diastolic calcium oscillations (m-SCR) occurred across most of the mapping field, at all pacing rates tested. Ryanodine (1 μM) eliminated all m-SCR activity. Low-dose caffeine (1 mM) increased m-SCR amplitude (+10.4 ± 4.4%, P < 0.05) and decreased m-SCR time-to-peak (-17.4 ± 6.7%, P < 0.05) and its temporal synchronization (i.e., range) across the mapping field (-26.9 ± 17.1%, P < 0.05). Surprisingly, carbenoxolone increased the amplitude of m-SCR activity (+14.8 ± 4.1%, P < 0.05) and decreased m-SCR time-to-peak (-11.3 ± 9.6%, P < 0.01) and its synchronization (-37.0 ± 19.1%, P < 0.05), similar to caffeine. In isolated myocytes, carbenoxolone (50 μM) had no effect on the frequency of aftercontractions, suggesting the effect of cell-to-cell uncoupling on m-SCR activity is tissue specific. Therefore, in the whole heart, overt m-SCR activity caused by calcium release from the SR can be induced over a broad range of pacing rates. Enhanced ryanodine receptor open probability and, surprisingly, decreased cell-to-cell coupling increased the amplitude and temporal synchronization of spontaneous calcium release in tissue.

Ryanodine Receptor is a Magnesium Channel: Consequences of Selectivity on Physiology

The ryanodine receptor (RyR) releases Ca2+ out of the sacroplasmic reticulum (SR) and is therefore thought of a calcium channel. However, the most abundant divalent cation is Mg2+ (present at ∼1 mM both in the cytoplasm and the SR) and RyR has a very high affinity for symmetrically applied Mg2+. This is seen in single-channel recordings when even 1 mM of symmetric Mg2+ reduces the current from 10 mM luminal Ca2+ by ∼25%. Here it is shown why changes in [Mg2+] (e.g., due to ischemia or exercise) disproportionately affect unitary Ca2+ current, in addition to changing RyR open probability. A recent model of ion permeation through RyR is used to first describe the mechanism behind this high Mg2+ affinity and RyR's weak K+ affinity and then analyze their effect on Ca2+ release. Loosely speaking, [K+] defines the SR membrane potential and [Mg2+] defines unitary Ca2+ current for a fixed SR membrane potential and SR Ca2+ load. Counterintuitively, while K+ occupancy in the pore is low, K+ still provides the vast majority of countercurrent during Ca2+ release. Moreover, the opposite is true for Mg2+; unless [Mg2+] is unusually high, Mg2+ provides little countercurrent despite having the largest number of ions in the pore.

Ca2+ wave probability is determined by the balance between SERCA2-dependent Ca2+ reuptake and threshold SR Ca2+ content

In this manuscript, we determined the roles of the sarcoendoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and the ryanodine receptor (RyR) in Ca(2+) wave development during β-adrenergic stimulation. SERCA2 knockout mice (KO) were used 6 days after cardio-specific gene deletion, with left ventricular SERCA2a abundance reduced by 54 ± 9% compared with SERCA2(flox/flox) controls (FF) (P < 0.05). Ca(2+) waves occurred in fewer KO than FF myocytes (40 vs. 68%, P < 0.05), whereas the addition of isoproterenol (ISO) induced waves in an equal percentage of myocytes (82 vs. 64%). SERCA2-dependent Ca(2+) reuptake was slower in KO (-ISO, KO vs. FF: 15.4 ± 1.2 vs. 21.1 ± 1.4 s(-1), P < 0.05), but equal during ISO (+ISO, KO vs. FF: 21.9 ± 3.3 vs. 27.7 ± 2.7 s(-1)). Threshold SR Ca(2+) content for wave development was lower in KO (-ISO, KO vs. FF: 126.6 ± 10.3 vs. 159.3 ± 7.1 µmol/L, P < 0.05) and was increased by ISO only in FF (+ISO, KO vs. FF: 131.7 ± 8.7 vs. 205.5 ± 20.4 µmol/L, P < 0.05). During ISO, Ca(2+)/calmodulin-dependent kinase II (CaMKII)-dependent phosphorylation of RyR in KO was 217 ± 21% of FF (P < 0.05), and SR Ca(2+) leak indicated higher RyR open probability in KO. CaMKII inhibition decreased Ca(2+) spark frequency in KO by 44% (P < 0.05) but not in FF. Mathematical modelling predicted that increased Ca(2+) sensitivity of RyR in KO could account for increased Ca(2+) wave probability during ISO. In ventricular cardiomyocytes with reduced SERCA2 abundance, Ca(2+) wave development following β-adrenergic stimulation is potentiated. We suggest that this is caused by a CaMKII-dependent shift in the balance between SERCA2-dependent Ca(2+) reuptake and threshold SR Ca(2+) content.

The effects of hydrogen peroxide on intracellular calcium handling and contractility in the rat ventricular myocyte

Elevations in reactive oxygen species are implicated in many disease states and cause systolic and diastolic myocardial dysfunction. To understand the underlying cellular dysfunction, we characterised the effects of H 2O 2 on [Ca 2+] i handling and contractility in the rat ventricular myocyte. This was achieved using patch clamping, [Ca 2+] i measurement using Fluo-3, video edge detection and confocal microscopy. All experiments were performed at 37 °C. 200 μM H 2O 2 resulted in a 44% decrease in the [Ca 2+] i transient amplitude, a 30% increase in diastolic [Ca 2+] i and an 18% decrease in the rate of systolic Ca 2+ removal. This was associated with a 61% reduction in systolic shortening, a contracture of 3 μm and a 42% increase in relaxation time respectively. The decrease in the [Ca 2+] i transient amplitude could be explained by a 27% decrease in SR Ca 2+ content. This, in turn results from a 22% decrease of SERCA activity. The decreased SR Ca 2+ content also provides a mechanism for a reduction in [Ca 2+] i spark frequency with no evidence for a Ca 2+ independent modification of ryanodine receptor open probability. We conclude that decreased SERCA activity is the major factor responsible for the changes of the systolic [Ca 2+] i transient.

Does Protein Kinase A–Mediated Phosphorylation of the Cardiac Ryanodine Receptor Play Any Role in Adrenergic Regulation of Calcium Handling in Health and Disease?

See related articles, pages 1743–1752 Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative …

Synchronized Spontaneous Calcium Release Events Throughout The Intact Heart

Intracellular calcium (Ca) dysregulation associated with cardiac disease (e.g. heart failure) has been linked to mechanisms of ventricular arrhythmias. Such arrhythmias can arise at the sub-cellular level from delayed afterdepolarizations that are due to spontaneous calcium release from the sarcoplasmic reticulum. The mechanisms that govern an aggregate of sub-cellular spontaneous calcium release events at the tissue level (i.e. an SCR) are not well understood. We hypothesize that in tissue, an SCR can be significantly influenced by ryanodine receptor (RyR) function. Methods: High resolution optical mapping of Ca (Indo-1AM) from the anterior surface of the Langendorff perfused guinea pig heart (n=4) was performed in hearts under high Ca conditions ([Ca2+]e=5.5mM), with and without caffeine (CAFF, 1mM) to enhance RyR open probability. Endocardial cryoablation was performed to eliminate Purkinje fibers and cytochalasin-D (7μM) was administered to remove motion artifact. Fifteen seconds of rapid pacing (400-160 ms cycle length) followed by a pause was used to induce SCR activity. Results: In all preparations, synchronized SCR activity was observed across the entire anterior surface of the heart with and without CAFF. SCR activity increased in magnitude and occurred earlier with decreasing pacing cycle length. With CAFF, the amplitude of SCR activity increased (+10.9%, p < 0.05) and occurred earlier (+15.6%, p < 0.05). CAFF also decreased the spatial heterogeneity of SCR onset across the mapping field (-37.4%, p < 0.05), suggesting that increased RyR open probability enhances the synchronization of SCR activity. Conclusions: These results demonstrate that sub-cellular spontaneous calcium release events (an SCR in tissue) occur over a broad region of the intact heart and are enhanced when RyR open probability is increased. SCR activity may be an important mechanism of arrhythmogenesis in heart disease associated with calcium dysregulation.